In recent times, there has been considerable research, clinical and commercial activity in the production and use of monoclonal antibodies for diagnostic and therapeutic purposes. Although the general technique of the fusion of spleen cells with myeloma cells was published by Kohler and Milstein in 1975, the production of monoclonal antibodies to specific antigens is still fraught with difficulties, especially where the antibodies must recognize and be able to distinguish between closely related antigens. Some variations of protein antigens differ from each other by a single amino acid residue, as is the case with many hemoglobin variants. Monoclonal antibodies directed to these protein antigens must be able to recognize and distinguish these proteins from each other on the basis of a single amino acid difference.
Human hemoglobin (Hb) is a protein about which a great deal of structural and functional information has become available. There are more than four hundred mutants or variants of hemoglobin known. Most of these variants differ from normal hemoglobin, hemoglobin A (Hb-A) by a single amino acio substitution. Human hemoglobin variants such as, for example, sickle-cell hemoglobin or hemoglobin S (Hb-S), as it is known in the art, and hemoglobin C (Hb-C) differ from normal hemoglobin A (Hb-A) by a single amino acid substitution at the sixth amino acid residue of the .beta.-globin chain. In Hb-S, valine is substituted for glutamic acid and in Hb-C, lysine is substituted for glutamic acid. These amino acid substitutions occur as tne result of single base changes in the globin gene of normal Hb-A. Efforts have been made to devise metnods which would identify and distinguish these variants from the normal protein.
Th. Papayannopoulou, T. C. McGuire, G. Lim, E. Garzel, P. E. Nute and G. Stamatoyannopoulos, in an article entitled "Identification Of Haemoglobin S In Red Cells And Normoblasts, Using Fluorescent Anti-Hb S Antibodies", (Brit. J. Haemat., 34, 25 (1976)) described procedures for the preparation of horse polyclonal antibodies monospecific for Hb-S and their use in detecting Hb-S in red cells and erythroid precursors.
G. Stamatoyannopoulos, D. Lindsley, Th. Papayannopoulou, M. Farquhar, M. Brice, and P. E. Nute, G. R. Serjeant and H. Lehmann, "Mapping Of Antigenic Sites On Human Haemoglobin By Means Of Monoclonal Antibodies And Haemoglobin Variants", The Lancet, Oct. 31, 1981, discloses the usefulness of variant hemoglobins in defining antigenic sites recognized by anti-globin monoclonal antibodies.
R. H. Jensen, W. Bigbee and E. W. Branscomb, in "Somatic Mutations Detected By Immunofluorescence And Flow Cytometry", (UCRL-88226, Lawrence Livermore National Laboratory, Livermore, Calif., Jan. 23, 1983) disclose immunofluorescence and flow cytometric techniques for the detection of certain somatic cell mutations, using both monoclonal and polyclonal antibodies.
The immunofluorescence and flow cytometric techniques disclosed by Jensen et al., (UCRL-88226), are based on a single-cell detection system for studying somatic cell mutations. The hemoglobin system, among several biochemical genetic systems in man, offers the best possibilities for use in these studies for various reasons. In addition to a great deal of structural and functional information about hemoglobin being readily available, and the easy availability of hemoglobin in large enough quantities for research purposes, ninety-five percent of the protein in erythrocytes is Hb, and about 20 to 50 percent of the protein per cell is abnormal in the case of most heterozygous structural mutations of the globin gene. Over 350 of hemoglobin mutational variants have been structurally characterized and are thus available for further mutation research. Most of these exhibit single amino acid substitutions indicating single base substitutions, although double substitutions, amino acid deletions, amino acid insertions and variants with elongated or shortened polypeptide chains are known. Most of these Hb variants are postulated to exist at very low frequencies in circulating red cells of every genetically normal individual. Furthermore, mature erythrocytes lend themselves very satisfactorily to single cell assays. They are easily obtained in large numbers and can be readily analyzed in suspension and express mutations that occur in the erythroid progenitors.
It would, therefore, be highly desirable to have an assay or method for the detection of heterozygous normal/mutant cells, i.e., cells which contain co-dominantly expressed genes in which one allele is normal and the other is mutant. Alleles are alternative forms of an individual gene. Such cells should produce both normal and mutant gene products in the case of neutral mutations and proteins coded for by only one allele in the case of "null" mutations. A "null" mutation is a single mutational event resulting in the lack of a functional gene product. Such a method could be either a clonogenic assay of mutant cells or an immunologic detection of cells which contain mutant gene products.